Visualization of Dendritic Spines

by Josef Spacek and John Fiala

Freeze Fracture Electron Microscopy

Although electron microscopy resolved the issue of whether dendritic spines were real, the method introduced its own complications. Preparation of a soft tissue specimen for transmission electron microscopy involves a difficult process. The tissue must be fixed, stained with heavy metals, dehydrated, and embedded in a compound such as epoxy that can be hardened for thin sectioning. The tissue must be cut into ultrathin sections for high resolution transmission electron microscopy. This sectioning precludes the direct observation of extensive cell surfaces or 3D structure.

Some of these problems were mitigated by the development (in the 1960's) of freezing methods of tissue preparation followed by electron microscopy. In the freeze-fracture method, the frozen tissue is fractured to produce a surface for investigation. The surface is plated with platinum and imaged in an electron microscope. The result is an image of the fractured surface, often revealing parts of dendritic spines.

Electron micrograph of a freeze fracture of a spine on a hippocampal pyramidal neuron.

Unfortunately, as with ultrathin section electron microscopy, the entire 3D structure of a dendritic spine along with its intracellular components cannot be readily determined from the imaged surface.

References

• Sandri C, Van Buren JM, Akert K (1977) Membrane Morphology of the Vertebrate Nervous System. A Study with Freeze-etch Technique. Progress in Brain Research, Volume 46, Elsevier Scientific: Amsterdam.
• Harris KM, Landis DMD (1986) Membrane structure at synaptic junctions in area CA1 of the rat hippocampus. Neuroscience 19:857-872.

Next: Serial Section Electron Microscopy and Three-Dimensional Reconstruction